Toll genes are associated with determination of dorsoventral axis in the embryogenesis of Drosophilia (e.g., see Cell 52, 269-279, 1988; Annu. Rev. Cell Dev. Biol. 12, 393-416, 1996), and with innate immunity detecting invading pathogens in adult body (e.g., see Nature 406, 782, 2000; Nat. Immunol. 2, 675, 2001; Annu. Rev. Immunol. 20, 197, 2002), and the Toll is a type I transmembrane receptor having Leucine-rich repeat (LRR) in extracellular domains. Among cytokines being signaling substances between cells that play an important role in immunoreaction and response during infection, hematogenesis, viral infection, and impairment of tumor cell, cytokine transmitting signals between lymphocytes is called Interleukin (hereinafter referred to as “IL”). It has been clarified that the intracytoplasmic domain of the type I transmembrane receptor is highly homologous with the intracytoplasmic domain of mammalian IL-1 receptor (IL-1R) (e.g., see Nature 351, 355-356, 1991; Annu. Rev. Cell Dev. Biol. 12, 393-416, 1996; J. Leukoc. Biol. 63, 650-657, 1998).
Recently, mammal homologue of Toll genes has been identified (e.g., see Nature 388, 394-397, 1997; Proc. Natl. Acad. Sci. USA 95, 588-593, 1998; Blood 91, 4020-4027, 1998; Gene 231, 59-65, 1999) and 10 members of human TLR family (TLR1-10) have been reported so far. The role of TLR family is to recognize discrete pathogen-associated molecular patterns (PAMPs) as a pattern recognition receptor (PRR) recognizing common bacterial structure, to trigger the activation of similar intracellular signaling pathway leading to nuclear translocation of a transcription factor, NF-κB. The signaling pathway ultimately culminates in the production of inflammatory cytokines to evoke host defense responses and further evoke host defense responses to acquired immunity. Moreover, various TLR ligands are reported recently.
TLR1 recognizes triacylated lipoproteins (e.g., see J. Immunol. 169, 10-14, 2002). TLR2 recognizes a variety of bacterial components such as peptidoglycan (PGN), bacterial triacylated lipoproteins, mycoplasmal diacylated lipoproteins, and GPI anchor of Trypanosoma cruzi (e.g., see Science 285, 732, 1999; Science 285, 736, 1999; J. Biol. Chem. 274, 33419, 1999; Immunity 11, 443, 1999; J. Immunol. 164, 554, 2000; Nature 401, 811, 1999; J. Immunol. 167, 416, 2001; Nat. Med. 8, 878-884, 2002). TLR3 is involved in recognition of double-stranded RNA, which is generated in the life cycle of RNA viruses (e.g., see Immunity 11, 443-451, 1999). TLR4 is a receptor for lipopolysaccharide (hereinafter LPS), a glycolipid specific to Gram-negative bacteria cell wall (e.g., see Nature 413, 732-738, 2001; J. Immunol. 162, 3749-3752, 1999). TLR5 recognizes flagellin, a protein component of bacterial flagella (e.g., see Science 282, 2085-2088, 1998). TLR6 is required for recognition of diacylated lipoproteins (e.g., see Nature 410, 1099-1103, 2001), whereas TLR7 is crucial for recognition of imidazoquinoline, an antiviral synthetic compound, and its derivative, R-848 (e.g., see Int. Immunol. 13, 933-940, 2001). TLR9 is a receptor for DNA with bacterial unmethylated CpG motif (5′-Pu-Pu-CpG-Pyr-Pyr-3′) (e.g., see Nat. Immunol. 3, 196-200, 2002).
Intracellular signaling pathways of TLRs are elicited from the TIR domain, which is conserved among the cytoplasmic regions of TLRs. A cytoplasmic molecule, MyD88, contains a TIR domain and a death domain. The death domain of MyD88 is required for interaction with other death domain-containing molecules such as IRAK-1 and IRAK-4 (e.g., see Nature 408, 740-745, 2000; Immunity 7, 837-847, 1997; Moll. Cell. 11, 293-302, 2003). The TIR domain is reportedly required for formation of dimers with other TIR domain-containing receptors or adaptors. Indeed, MyD88-deficient mice show neither splenocyte proliferation nor production of proinflammatory cytokines in response to all TLR ligands and IL-1, suggested that MyD88 is essential for the immune responses of all TLRs and the IL-1 receptors (e.g., see Immunity 9, 1, 143-150, 1998). However, a TLR3 ligand, poly (I:C), and a TLR4 ligand, LPS, still stimulate the expression of specfic genes such as IFN-β, in MyD88-deficient mice. Induction of IFN-β expression leads to maturation of dendritic cells and subsequent expression of IFN-inducible genes (e.g., see J. Immunol. 167, 5887-5894, 2001; J. Immunol. 166, 5688-5694, 2001). These observations suggested that TLR signaling is composed of at least two pathways: a MyD88-dependent pathway that leads to production of proinflammatory cytokines, and a MyD88-independent pathway associated with induction of IFN-inducible genes and maturation of dendritic cells. Moreover, the specificity of the MyD88-dependent signaling pathways through all TLRs is provided by TIRAP, the second TIR domain-containing adaptor discovered (e.g., see Nat. Immunol. 2, 835-841, 2001; Nature 413, 78-83, 2001). TIRAP-deficient mice show severe defects in activation of the MyD88-dependent signaling pathway through TLR2 and TLR4, but not other TLRs (e.g., see Nature 420, 324-329, 2002; Nature 420, 329-333, 2002).
Although the precise molecular mechanisms of the MyD88-independent signaling pathways are unknown, identification of another TIR domain-containing molecule, TRIF (e.g., see J. Immunol. 169, 6668-6672, 2002; Nat. Immunol. 4, 161-167, 2003), and genetic evidence from mice carrying a mutation in this gene revealed that TRIF plays a crucial role in MyD88-independent signaling pathway shared by TLR3 and TLR4 (e.g., see Science 301, 640-643, 2003; Nature, 424, 743-748, 2003). Furthermore, recent studies showed that two noncanonical IκB kinases (IKKs), that is, IKK-ι/IKKε and TBK1/T2K, interact with TRIF, activate IRF-3 and, finally, lead to IFN-β induction (e.g., see Science 300, 1148-1151, 2003; Nat. Immunol. 4, 491-496, 2003).
To date, two more kinds of TIR domain-containing adaptors have been identified in the human genome. One is called SARM (abbreviation for SAM and ARM domain-containing protein), whose physiological function in the TLR/IL-1R signaling remains unclear (e.g., see Genomics 74, 234-244, 2001, Trends Immunol. 24, 286-290, 2003). The other is TRAM (abbreviation for TRIF-Related Adaptor Molecule, also called TIRP) (e.g., see J. Biol. Chem. 278, 24526-24532, 2003). Previous in vitro analysis indicated that ectopic expression of TRAM activates NF-κB, as does MyD88, TIRAP and TRIF. However, it did not activate the IFN-β promoter unlike TRIF. Dominant negative mutants of this protein inhibit the NF-κB activation through IL-1R, but not through TLRs. This indicated that TRAM is a specific adaptor protein in the IL-1R-mediated MyD88-dependent signaling pathway. However, the role of TRAM in vivo remains to be clarified.
The object of the present invention is to provide a non-human animal model non-responsive especially to endotoxin being Gram-negative bacteria cell wall fraction, which is useful to elucidate the function of TRAM, a TIR domain-containing TRIF-related adaptor protein; and a method for screening substances promoting or suppressing responses to ligands recognized by TLR4 with the use of the non-human animal models non-responsive to endotoxin, and the like.
The present inventors generated TRAM-deficient mice, analyzed the role of TRAM in the TLR/IL-1R signaling pathway in vivo and obtained the below knowledge.
TRAM-deficient mice showed severe defects in cytokine production, splenocyte proliferation and up-regulation of surface molecules, in response to the TLR4 ligands, but not to other TLR ligands. Furthermore, TLR4-mediated, but not TLR3-mediated, expression of IFN-β and IFN-inducible genes was inhibited in TRAM-deficient mice. In intracellular signaling, LPS-induced autophosphorylation of IRAK-1 and the early phase of NF-κB activation were intact in TRAM-deficient mice. However, no activation of IRF-3 was observed while a defect in the late phase of NF-κB activation in response to LPS, but not to poly (I:C), was observed in TRAM-deficient cells. Given that the latter event is a feature of the MyD88-independent signaling pathway, it was indicated that TRAM specifically mediates the MyD88-independent pathway of TLR4 signaling.
In TRAM-deficient mice, TLR4-mediated activation of the MyD88-dependent pathway, which is characterized by autophosphorylation of IRAK-1 and the early phase of NF-κB activation, was comparable to that of wild-type cells. However, TLR4-mediated production of proinflammatory cytokines was reduced. Similarly, TLR4-mediated production of proinflammatory cytokines was significantly reduced in mice lacking TRIF, which is essential for TLR4- and TLR3-mediated MyD88-independent pathways. As MyD88-deficient mice showed similar phenotype, activation of the MyD88-independent pathway is clearly required for induction of proinflammatory cytokines (e.g., see Science 301, 640-643, 2003, Identification of Lps2 as a key transducer of MyD88-independent TIR signaling; Nature, 424, 743-748, 2003). Therefore, in TLR4 signaling, activation of both MyD88-dependent and MyD88-independent (TRAM/TRIF-dependent) pathways is required for proinflammatory cytokine production. However, in signaling pathways of TLR2, TLR5 and TLR9, none of which activate the MyD88-independent pathway, only the activation of MyD88-dependent pathway is sufficient to induce proinflammatory cytokines (e.g., see Science 282, 2085-2088, 1998; Nat. Immunol. 3, 392-398, 2002; J. Exp. Med. 192, 595-600, 2000; Curr. Biol. 10, 1139-1142, 2000). Therefore, at present, it remains unclear why TLR4 signaling requires activation of both MyD88-dependent and TRIF-dependent pathways to induce proinflammatory cytokines. However, it has become clear that only TLR4 utilizes all of the presently characterized TIR domain-containing adaptors, that is, MyD88, TIRAP, TRIF and TRAM.
As described above, TRAM-deficient mice showed normal responses to ligands for TLR2, TLR7, TLR9 and IL-1β, but severely defective MyD88-dependent responses to the ligands recognized by TLR4. Furthermore, activation of the TLR4-mediated MyD88-independent, but not MyD88-dependent, signaling cascade was abolished in TRAM-deficient mice. Although this phenotype was reminiscent of that of TRIF-deficient mice which lack activation of MyD88-independent pathway in both TLR3 and TLR4 signaling, TRAM-deficient mice showed a normal response to TLR3 ligands. These results indicate that TRAM is an adaptor molecule that provides specificity for the MyD88-independent pathway of TLR4 signaling.
The present invention has been thus completed based on the above knowledge.